Controlled release compositions for interferon based PEGT/PBT block copolymers and method for preparation thereof

ABSTRACT

The invention discloses a pharmaceutical composition for the controlled release of relatively toxic active compounds, in particular for bioactive proteins from the class of interferons. The composition comprises a biodegradable block copolymer constructed from poly(ethylene glycol) terephthalate (PEGT) and poly(butylene terephthalate) (PBT). The composition is provided in the form of injectable microparticles, of an injectable liquid which may have self-gelling properties, or of a solid implant. The invention further provides a pharmaceutical kit comprising the composition, methods for preparing the composition, and the pharmaceutical uses relating thereto.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/466,430, filed May 15, 2009; which is a continuation of Ser. No.11/774,107, filed Jul. 6, 2007; which is a continuation of PCTapplication no. PCT/NL2006/000006, designating the United States andfiled Jan. 6, 2006; which claims the benefit of the filing date ofEuropean application no. EP 05075043.9, filed Jan. 7, 2005; each ofwhich is hereby incorporated herein by reference in its entirety for allpurposes.

FIELD

The present invention relates to pharmaceutical compositions for thecontrolled release of active compounds. The compositions are in the formof polymeric microparticles, in-situ gels, or solid implants. They arebased on biodegradable polymers and are particularly useful for thecontrolled delivery of therapeutic proteins or peptides. Moreover, theinvention relates to polymeric microparticles comprised in thecompositions and to methods of making such microparticles. In furtheraspects, the invention relates to pharmaceutical kits which comprise thecompositions, and to the uses of such kits.

BACKGROUND

Parenteral dosage forms with slow drug release properties have beendeveloped to answer the need for improving the therapeutic use of drugsubstances which should not be administered orally due to theirphysicochemical properties, and which have a relatively short half lifebecause of which they have to be injected frequently. Frequentinjections are uncomfortable to patients, and if the injections have tobe given by physicians or nurses, they are also rather costly. Theexperience of discomfort and pain may result in patient incompliance andjeopardize the success of the therapy.

The number of drug substances which cannot be administered by theconvenient oral route is presently increasing, primarily as aconsequence of the recent advances of biotechnological research in thepharmaceutical area, which has lead to an increased number of highlypotent peptide and protein drugs. Perhaps with the exception of somesmaller peptides, however, these compounds are relatively unstable ingastrointestinal fluids and, more importantly, too large and hydrophilicas molecules to become absorbed through the intestinal mucosa to asubstantial extent. For some of these drug substances, injectable orimplantable controlled release formulations are being developed in orderto lower the dosing frequency and thus reduce patient discomfort, andachieve a higher level of compliance and therapeutic success.

Parenteral controlled release dosage forms are usually in the form ofmacroscopic, solid single- or multiple-unit implants (such as polymericrods and wafers), microparticle suspensions, and more recently alsogels, including in-situ forming gels. Drug-loaded solid implants areavailable as non-degradable polymeric, ceramic or metal devices whichhave to be surgically removed after the designated period of drugaction, or as biodegradable polymeric forms which require no removal. Anexample for a non-degradable implant is Bayer's VIADUR®, which releasesthe peptide drug, leuprolide, over a period of one year. An example fora biodegradable implant is AstraZeneca's ZOLADEX®, which is a polymericrod capable of releasing the peptide drug, goserelin, over periods ofone and three months, respectively.

Shortly after the market introduction of the first biodegradableimplants, controlled release microparticles became available, such asTakeda's Lupron® Depot formulations, which release leuprolide overperiods of one, three, and four months, respectively. In order to injectsuch microparticles, they have to be suspended in an aqueous carrier.For stability reasons, however, depot microparticles cannot usually bestored as an aqueous suspension, but they have to be reconstituted froma dry powder.

Various designs of drug-loaded microparticles and methods for theirpreparation are described in E. Mathiowitz et al., Microencapsulation,in: Encyclopedia of Controlled Drug Delivery (ed. E. Mathiowitz), Vol.2, pp. 493-546, John Wiley & Sons (1999), which is incorporated hereinby reference.

To enable the injection of drug delivery systems through particularlyfine needles to provide increased patient comfort, drug deliveryscientists have in recent years begun to develop injectable gels whichare capable of forming subcutaneous or intramuscular depots. In one ofthe concepts, gel formulations are designed which are highly shearthinning and thixotropic. By applying shear force prior toadministration, the viscosity of these gels is substantially reduced,allowing for injection with a relatively small needle, whereas the gelstrength is recovered slowly after administration. According to anotherconcept, liquid compositions are formulated which, after administration,form gels in response to changes of their environment, such as pH,temperature, ionic strength. According to a third approach, liquidpolymer formulations comprising a non-aqueous solvent are injected. Uponadministration, the solvent diffuses away from the injection site, whichleads to the precipitation of polymeric particles or to the formation ofa gel.

Biodegradable injectable gels have been discussed in detail by A. Hatefiet al., Journal of Controlled Release (2002) 80:9-28, which document isincorporated herein by reference.

The therapeutic usefulness of several polymeric carriers for controlledrelease, in particular that of polymers and copolymers of lactic acidand glycolic acid, has been demonstrated for a few active compounds,such as leuprolide, goserelin, buserelin, and triptorelin, which are allpeptides with a very large therapeutic index, i.e. having a very lowtoxicity even at levels far above the therapeutically effectiveconcentrations. In contrast, less tolerable active compounds such aserythropoietin and interferons, whose precisely controlled delivery isnecessary for achieving therapeutic effects without intolerable sideeffects, have not successfully been developed as controlled releasedosage forms. A major difficulty is that the biodegradable polymericcarriers used in the successful earlier product are apparently notcapable of providing zero-order or nearly zero-order release profiles.Instead, they produce highly undesirable initial burst release uponadministration. Furthermore, the autocatalytic degradation of polymersand copolymers of lactic acid and glycolic acid may also lead to dosedumping effects at later stages of drug release. On the other hand,other new polymers which have been discussed as improved controlledrelease carriers for therapeutic compounds do not have the safety recordof the poly(lactides) and poly(glycolides).

Thus, there is a need for new polymeric delivery systems which haveproven biocompatibility, but which are also capably of bettercontrolling the release of relatively toxic therapeutic compounds thanthe previously used carriers.

It is therefore an object of the invention to provide new controlledrelease compositions comprising one or more polymeric carriers havingexcellent biocompatibility and a relatively toxic therapeutic compoundwhich should not be given via the oral route, such as a protein.

Another object of the invention is to provide microparticles, implants,and gel compositions comprising an active compound which is released ata controlled rate. Furthermore, it is an object of the invention toprovide kits which contain such compositions and pharmaceutical usesthereof. Further objects will become apparent on the basis of thefollowing description and patent claims.

SUMMARY

The invention provides a pharmaceutical composition for the controlledrelease of interferons. More specifically, the composition provided bythe invention comprises a biodegradable polymer and an active compoundselected from the group of interferons. The biodegradable polymer is ablock copolymer constructed from poly(ethylene glycol)-terephthalate(PEGT) and poly(butylene terephthalate) (PBT). A preferred activecompound is an interferon selected from the family of alfa-interferons.

In a further embodiment, the composition of the invention is designed tocomprise microparticles which contain the block copolymer and at leastsome of the interferon comprised in the composition. Such composition isparticularly useful as a parenteral controlled release formulation whichcan be injected intramuscularly or subcutaneously.

In another embodiment, the invention provides a pharmaceutical kitcomprising a first and a second sealed compartment, wherein the firstcompartment comprises such microparticle-based composition insubstantially dry form, and wherein the second compartment comprises anaqueous liquid carrier for reconstituting the composition into aninjectable microparticle suspension.

In a further embodiment, the composition of the invention is shaped as asolid implant.

Further embodiments include methods for the preparation of thecomposition and the pharmaceutical uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the release of interferon from copolymermicroparticles in vitro and in hamsters.

FIG. 2 represents the release of interferon from copolymermicroparticles in vitro and in monkeys.

DETAILED DESCRIPTION

In the discovery process leading to the present invention, it has beenfound that many of the polymers which have been suggested as controlledrelease agents for active compounds, such as polymers of lactic and/orglycolic acid or, are not very suitable for the delivery of relativelytoxic active compounds such as interferons. In particular, the releasebehavior appeared to be poorly controllable, especially when thepolymers are formed as microparticles or gels. For example, it seemsdifficult to avoid the so-called burst release, i.e. a rapid release ofa significant fraction of the incorporated active compound soon afteradministration, when using the conventional polymeric carriers.Depending on the therapeutic index of the respective active compound,such burst release can produce rather toxic effects in patients.

In contrast, it was surprisingly found that block copolymers of PEGT andPBT are capable of incorporating and releasing the interferons (abovementioned compounds) in a much better controlled fashion, with little orsubstantially absent burst effect, as will be further discussed in thisdescription.

Therefore, the invention provides a pharmaceutical composition forcontrolled release comprising a biodegradable polymer and an activecompound selected from the group of interferons, wherein thebiodegradable polymer is a block copolymer constructed frompoly(ethylene glycol)-terephthalate (PEGT) and poly(butyleneterephthalate) (PBT).

It has also been found by the inventors that the block polymersdescribed above can form a surprisingly suitable matrix forincorporating interferons for controlled release applications. Inparticular, they can incorporate large amounts of interferons withoutloss of bioactivity.

Another reason why the specified copolymers are particularly suitable isthat they are able to control the release of incorporated interferonsover a wide range of release profiles which may be considered desirable,depending on the specific therapeutic application. The polymeric carriermay be developed into various dosage form designs, such asmicroparticles, films, gels, and solid implants, and can involve a rangeof molecular weights and degrees of hydrophilicity which can—togetherwith the geometry of the dosage form or a unit thereof—achieve variousdurations of interferon release, and various types of release profiles.

A pharmaceutical composition is defined as a composition which istypically used for therapeutic or diagnostic purposes, or for wellnessand disease prevention. While many pharmaceutical compositions aredesigned and formulated for the immediate release of incorporated activecompounds, there are also compositions which possess controlled releasecharacteristics in order to provide an extended duration ofeffectiveness. Several terms have been used to describe various types ofcontrolled release characteristics. As used herein, controlled releaserefers to any modified active compound release such as delayed release,prolonged release, constant or zero-order release, extended release,sustained release, slow release, biphasic release etc.

The composition comprises a biodegradable polymer. According to theIUPAC terminology, a polymer is defined as a substance composed ofmacromolecules. In turn, a macromolecule is a molecule of high relativemolecular mass, the structure of which essentially comprises themultiple repetition of a number of constitutional units. In commonlanguage, however, the distinction between a polymer and themacromolecules which it comprises is not always made. This is also truefor the present description, which may attribute features to the polymerwhich should—strictly speaking—be attributed to macromolecules.

Biodegradability may be defined as the ability of a substance to bechemically degraded at physiological conditions, in physiologicalenvironments, or through enzymatic action. In the context of theinvention, it is preferred that the biodegradable polymer is degradablein a physiological environment—such as in physiological fluids at bodytemperature—even in the absence of enzymes in the sense that substantialdegradation occurs within the course of hours, days, weeks, months, oryears. The degradation may include various chemical mechanisms includinghydrolysis or oxidation. For the avoidance of misunderstandings,biodegradability does not mean that the biodegradable polymer mustdegrade into the respective monomeric units. It is sufficient that thedegradation process leads to soluble molecular species which can beeliminated from an organism by processes such as renal or hepaticexcretion. In the present invention, the polymer typically serves ascarrier for the active compound and as release controlling agent.

Furthermore, the biodegradable polymer is selected from the group ofblock copolymers constructed from poly(ethylene glycol) terephthalate(PEGT) and poly(butylene terephthalate) (PBT). A copolymer is defined asa polymer derived from more than one species of monomer. In a blockcopolymer (or block polymer), the constituent macromolecules haveadjacent blocks that are constitutionally different, i.e. adjacentblocks comprise constitutional units derived from different species ofmonomer or from the same species of monomer but with a differentcomposition or sequence distribution of constitutional units. A blockmay be defined as a portion of a macromolecule which comprises amultiple number of constitutional units that have at least one featurewhich is not present in the adjacent portions.

A number of block copolymers comprising PEGT and PBT have been describedin prior art, for example by J. M. Bezemer et al. (J. Control Release1999, 62 (3), 393-405; J. Biomed. Mater. Res. 2000, 52 (1), 8-17; J.Control Release 2000, 66 (2-3), 307-320; Control Release 2000, 67 (2-3),249-260; J. Control Release 2000, 67 (2-3), 233-248; J. Control Release2000, 64 (1-3), 179-192), R. Dijkhuizen-Radersma et al. (Biomaterials2002, 23 (24), 4719-4729; J. Biomed. Mater. Res. 2004, 71A (1), 118;Biomaterials 2002, 23 (6), 1527-1536; Pharm. Res. 2004, 21 (3), 484-491;Int. J. Pharm. 2002, 248 (1-2), 229-237; Eur. J. Pharm. Biopharm. 2002,54 (1), 89-93), and J. Sohier et al. (J. Control Release 2003, 87 (1-3),57-68; Eur. J. Pharm. Biopharm. 2003, 55 (2), 221-228), and in WO93/21858, EP 0 830 859 A2, and EP 1 090 928 A1, all of which documentsare incorporated herein in their entirety.

These copolymers can be understood as being composed of repeating blocksof hydrophilic poly(ethylene glycol) (PEG) and hydrophobic poly(butyleneterephthalate) (PBT). These poly(ether ester)s are typically prepared bypolycondensation of PEG, butanediol and dimethyl terephthalate.Alternatively, they can be understood as being composed of repeatingblocks of poly(ethylene glycol) terephthalate (PEGT) and PBT. Thesecopolymers usually have the properties of thermoplastic elastomers. Inan aqueous environment, they form hydrogels or hydrogel-like polymericnetworks, in which the polymer chains are not chemically but physicallycrosslinked. It is believed that the crosslinking is effected by theassociation of “hard” PBT segments into crystalline domains, whereasamorphous regions comprising “soft” PEG segments and some PBT areresponsible for the swelling behavior in water. In contrast to chemicalcrosslinks, these physical crosslinks are reversible at elevatedtemperatures or in appropriate solvents.

According to the invention, the active compound is selected from thegroup of interferons. Interferons represent a family of naturallyoccurring proteins derived from human cells and involved in variousfunctionalities of the immune system, such as in fighting viralinfections. Several interferons have been developed into pharmaceuticalproducts and are today available as products of genetic engineering foruse in the treatment of leukaemias, hepatitis, multiple sclerosis, andother severe diseases.

In contrast to several other active peptides and proteins which havebeen successfully developed into controlled release formulations,interferons have a relatively small therapeutic index. In other words,they show substantial toxicity at levels above the therapeuticallyeffective concentrations. Thus, their precisely controlled delivery isnecessary for achieving therapeutic effects without intolerable sideeffects.

One of the major classes of interferons is that of the alfa-interferons(IFN-alfa or IFN-alpha). Alfa-interferons comprise a number of nativeand modified proteins with similar molecular weight and functionality(see D. J. A. Crommelin et al., Pharmaceutical Biotechnology, HarwoodAcademic Publishers (1997), 219-222). Leukocytes are one of the majororigins of these proteins in humans. At least 23 different nativesubtypes and several modified versions of IFN-alfa are known, some ofwhich are available in pharmaceutical products. For example, a mixtureof several native IFN-alfa subtypes derives from pooled infected humanleukocytes has been commercially developed. The presently most importantmembers of the IFN-alfa group are the recombinant variants ofIFN-alfa-2a and IFN-alfa-2b. Another recombinant IFN-alfa used intherapy is IFN-alfacon-1.

The basic function of these interferons is the upregulation of theimmune system, such as the stimulation of immunological cells capable ofrecognizing and directly or indirectly destroying cancer cells orviruses. Among the therapeutic indications for alfa-interferons are(chronic) hepatitis B, (chronic) hepatitis C, hairy cell leukaemia,(chronic) myelogenous leukaemia, multiple myeloma, follicular lymphoma,carcinoid tumor, malignant melanoma, genital warts, bladder carcinoma,cervical carcinoma, renal cell carcinoma, laryngeal papillomatosis,mycosis fungeoides, condyloma acuminata, SARS, and (AIDS-related)Kaposi's sarcoma. In fact, it is presently most preferred according tothe invention that the active compound is selected from the group ofalfa-interferons.

The native members of the alfa-interferons have molecular masses between19-26 kDa and consist of proteins with lengths of 156-166 and 172 aminoacids. All IFN-alpha subtypes possess a common conserved sequence regionbetween amino acid positions 115-151 while the amino-terminal ends arevariable. Many IFN-alpha subtypes differ in their sequences at only oneor two positions. Naturally occurring variants also include proteinstruncated by 10 amino acids at the carboxy-terminal end.

Another major class of interferons is that of beta-interferons(IFN-beta), the presently most important representatives in therapybeing IFN-beta-1a and IFN-beta-1b. These interferons are used e.g. inthe management of certain forms of multiple sclerosis, in particularrelapsing forms of multiple sclerosis, to slow the accumulation ofphysical disability and decrease the frequency of clinicalexacerbations. Patients with multiple sclerosis in whom efficacy hasbeen demonstrated include patients who have experienced a first clinicalepisode and have MRI features consistent with multiple sclerosis.

Another therapeutically used class of interferons is that ofgamma-interferons (IFN-gamma). These interferons have antiviral,antiproliferative and immunomodulatory activities. One member of thegamma-interferons, IFN-gamma-1b, is currently marketed for themanagement of serious infections associated with chronic granulomatousdisease.

More recently, several additional classes of interferons were discoveredand described, including IFN-epsilon, IFN-kappa and IFN-lambda (see P.Kontsek et al., Acta Virol. 2003, 47 (4):201-15).

In particular, a composition according to the invention wherein theinterferon is selected from the group of alfa-interferons, andpreferably from the group consisting of IFN-alfa, IFN-alfa-2a,IFN-alfa-2b, IFN-alfacon-1, pegylated IFN-alfa-2a, pegylatedIFN-alfa-2b, truncated IFN-alfa-2a, truncated IFN-alfa-2b, fusionproteins of IFN-alpha and albumin, and a functional derivative thereofgives very good properties. In this context, an alfa-interferon may alsorepresent a mixture of various alfa-interferon variants, such as amixture of native alfa-interferons which are difficult or unnecessary toseparate and purify. The interferon may be extracted from livingorganisms or isolated cells or cell cultures. The cells and/or organismsfrom which the interferon is obtained may be modified, such as byinfection, in order to produce the desired interferon.

A composition according to the invention wherein the interferon is arecombinant interferon produced from genetically engineered cells ororganisms, wherein the cells or organisms are preferably selected frommammalian, insect, bacteria, yeasts, fungi and higher plant cells ororganisms, gives especially good properties.

One of the particularly suitable interferons for carrying out theinvention is a truncated version of IFN-alfa-2b or, optionally, amixture of more than one truncated versions of IFN-alfa-2b. For example,molecules comprising the IFN-alfa-2b amino acid sequence in which thelast 5 to 10 amino acids of the N-terminus have been deleted can beprepared by the currently available methods of genetic engineering. In afurther embodiment, variants of IFN-alfa-2b which are truncated by 7 or8 N-terminal amino acids are preferred.

It is preferred that the composition of the invention exhibits a releaseof the active compound over a period of at least about 7 days. Morepreferably, the interferon is released over at least about 10 days, orat least about 14 days. In further embodiments, the release occurs overat least about 3 weeks, 4 weeks, 6 weeks, and 2 months, respectively.Presently much preferred is a release over a period of about 10 days to1 month. Which grade of polymer should be selected and which furtherspecific features are useful for achieving such duration of releasedepends at least partially on the selected dosage form design and isdescribed in further detail below.

The invention also relates to a pharmaceutical composition forcontrolled release comprising a biodegradable polymer and one or moreactive compounds selected from the group of interferons, wherein atleast about 80 wt. % of the active compound, based on the total weightof the active compound, is released in monomeric, non-aggregated form.In accordance with this embodiment of the invention, the biodegradableis preferably, but no limited to, a block copolymer as defined herein,constructed from poly(ethylene glycol) terephthalate (PEGT) andpoly(butylenes terephthalate) (PBT). It has been found by the inventorsthat the block polymers described above can form a surprisingly suitablematrix for incorporating interferons for controlled releaseapplications. In particular, they can incorporate large amounts ofinterferons without loss of bioactivity, and they appear to preserve themonomeric, non-aggregated state of the incorporated interferons. This isremarkable as interferons are known to be sensitive to various polymersand process conditions, and particularly liable to aggregation which isoften associated with deactivation. In contrast, using the blockpolymers specified herein as carriers for interferons, it is possible toachieve that most of the incorporated interferon is released inmonomeric form.

Preferably, the polymer grade and the processing conditions should beselected to ensure that at least about 80% of the incorporated activeingredient, i.e. interferon, is released in monomeric, non-aggregatedform. Even more preferably, at least about 90% of the interferon isreleased as monomers, or according to further embodiments, at leastabout 95%, 97%, and 98%, respectively. These percentages are by weight,based on the total weight of the incorporated active ingredient.

Further preferred forms of the compositions according to the inventionare described below.

A unit dose of the composition, which is that amount of the compositionwhich is administered at a time, preferably comprises an amount ofactive compound which is equivalent to 1 million international units(MIU) of the respective interferon. The exact amount which isincorporated depends, of course, on the release profile of thecomposition and on the daily or weekly dose which a particular patientshould receive.

In one of the embodiments, the composition is adapted to release atleast about 5 MIU of interferon over 14 days, i.e. over the first 14days after administration. In another embodiment, it comprises a dose ofabout 10 to about 150 MIU, which dose is released over a period of about10 days to about 1 month, in particular over a period of about 14 days.Also preferred is a composition which comprises and releases, over thesame period of time, a dose of about 20 to about 100 MIU. Suchcompositions are particularly preferred if the active ingredient is analfa-interferon, such as IFN-alfa-2b or a derivative thereof.

Calculated for an average day within the period of interferon releaseafter administration, the composition is preferably adapted to releasean amount of about 0.5 to 20 MIU of the respective interferon, or fromabout 1 to 10 MIU. Depending on the shape of the release profile, it ispossible that the amount of active ingredient released within the firstday after administration is higher than 10 or 20 MIU, but the averagedaily release may still be in the preferred ranges.

The composition of the invention may be designed, formulated andprocessed so as to be suitable for a variety of therapeutic uses andmodes of administration, such as topical, oral, rectal, vaginal, orophthalmic administration; preferably, however, it is adapted forparenteral administration. As used herein, parenteral administrationincludes any invasive route of administration, such as subdermal,intradermal, subcutaneous, intramuscular, locoregional, intratumoral,intraperitoneal, interstitial, intralesional, with some less preferencein the context of this invention also intravenous, intraarterial etc.Highly preferred routes of administration of the composition aresubcutaneous and intramuscular injection or implantation.

Being suitable for parenteral administration means in particular thatthe composition preferably is sterile and complies with the requirementsof the current pharmacopoeias with regard to the content of endotoxins,osmolality, etc. The excipients are preferably selected to be safe andtolerable for parenteral administration. In a further aspect, thecomposition is formulated to be relatively isotonic (or isoosmotic),such as in the region of about 150 to 500 mOsmol/kg, and preferably inthe region of about 250 to 400 mOsmol/kg. Furthermore, the pH should beapproximately in the physiological range in order to avoid pain andlocal intolerance upon injection. Preferably, the pH of the compositionis in the region of about 4 to 8.5, and more preferably in the region ofabout 5.0 to 7.5.

The composition of the invention may be designed and formulated so as tocomprise microparticles which in turn comprise the biodegradable blockcopolymer and the active compound, or at least a substantial fraction ofthe active compound present in the composition. In this case, the dosageform of the composition which is administered is typically an injectablesuspension comprising the microparticles and a liquid coherent carrier.

In the context of the invention, microparticles should be understood assolid or semisolid particles having a diameter in the region of about0.1 to about 500 μm, regardless of their shape or internal structure.For example, microparticles would also encompass microspheres andmicrocapsules. In a more preferred embodiment, the microparticles have adiameter from about 1 to about 300 μm. Moreover, it has been found thatdesirable release properties are best achieved for interferons that areincorporated into microparticles based on PEGT/PBT block copolymerswhich have a volume-average diameter of about 25 to about 200 μm, asmeasured by photon correlation spectroscopy. Selecting such particlesize will also ensure that a suspension of these microparticles iswell-syringeable and can be easily and conveniently administered viaintramuscular or subcutaneous injection.

Within this size range, the diameter may be further optimized forspecific product applications or to accommodate specific interferons.For example, in the case of—optionally truncated—interferon-alfa-2a andinterferon-alfa-2b it is presently most preferred to selectvolume-average microparticle diameters from about 30 to about 175 μm. Infurther preferred embodiments, the average diameter is in the range fromabout 50 to about 150 μm.

Preferably, the microparticles should have a relatively low porosity. Inparticular, it was found that desired release profiles for controlledrelease applications of alfa-interferons can be best accomplished whenthe presence of larger pores is largely avoided. In this context, largerpores may be defined as pores having a diameter of about 5 μm or more.Thus, in one of the preferred embodiments the majority of themicroparticles are substantially free of pores having a diameter ofabout 5 μm or more. In another embodiment, the majority of themicroparticles are substantially free of pores having a diameter ofabout 2 μm or more.

Optionally, the microparticles may be coated with a drug-free layer ofpolymer. Such embodiment may be useful to prevent an initial burstrelease of the incorporated active compound, or even achieve apre-determined lag-time until release begins, if so desired.

The microparticles are based on the block copolymer of PEGT and PBT,which is used as carrier and controlled release agent. It has beenfound, however, that not all copolymers of PEGT and PBT are equallyuseful to make microparticles for the controlled release of allinterferons. Furthermore, the intended release time or duration ofeffect is important for the selection of the block copolymer. In thecase of the alfa-interferons, it has been found that the copolymershould preferably comprise from about 50 to about 95 wt.-% PEGT, andconsequently from about 5 to about 50 wt.-% PBT. In another embodiment,the copolymer comprises from about 70 to about 95 wt.-% PEGT. Accordingto a yet further preferred embodiment, the copolymer contains from about70 to about 85 wt.-% PEGT.

To further specify the chemical composition of the copolymer, themolecular weight of the PEG segments of the PEGT component is animportant parameter. It has been found that alfa-interferons are veryreadily incorporated into copolymer microparticles whose release profilecan be adjusted within useful ranges when the average molecular weightof the PEG is from about 600 to about 3,000. Even more preferably, theaverage molecular weight of the PEG is from about 1,000 to about 2,000.

The selection of the average molecular weight of the PEG may also takethe average particle size into consideration. If, for example, arelatively small particle size is selected, e.g. for processing reasons,such as below about 100 μm, or even below about 75 μm, it is preferredto select a block polymer with a relatively low degree ofhydrophilicity, i.e. having a relatively low average molecular weight ofthe PEG, such as about 1,500 or less, or about 1,000 or less, especiallyif a duration of release of two weeks or longer is desired.Alternatively, or additionally, a low degree of hydrophilicity may alsobe achieved by selecting a relatively low content of PEGT segments, suchas not more than about 75 wt.-%.

Conversely, there may be reasons to select a relatively large averageparticles size, such as above about 100 μm, e.g. based on processingconsiderations or to achieve a desired in vivo behavior. In this case,it is presently preferred to select an average molecular weight of thePEG of about 1,000 to about 3,000, or of at least about 1,500, and/or arelatively high content of PEGT, such as at least about 75 wt.-%.

Furthermore, it may be useful to combine two or more different PEGT/PBTblock copolymers for preparing microparticles having an optimizedrelease behavior. The two or more block copolymers may differ, forexample, in their relative PEGT content, or they may differ in theaverage molecular weight of the PEG, or they may differ in bothparameters. For example, useful polymer blends for making themicroparticles with alfa-interferon as active agents may comprise twopolymers both having a PEGT content of about 80 wt. %, but with averagePEG molecular weights of about 1,000 and about 2,000, respectively.Another useful blend comprises two polymers having a PEGT content ofabout 80 wt.-% and an average PEG molecular weight of about 1,000 andabout 1,500, respectively. The two or more different polymers can beblended in various ratios, such as 50:50, 75:35, or 75:25.

It has been found that the compositions of the invention are suitablefor incorporating alfa-interferons and achieving release times of about1 to about 8 weeks. For example, by choosing appropriate blockcopolymers, the release profile may be adjusted to provide drug effectsover periods from about 10 days or 2 weeks to about 4 weeks, which isthe presently most preferred release time. The release time, or durationof release, should be understood as the time in which at least about 80wt.-%, and more preferably at least 90 or 95 wt.-% of the incorporatedactive compound are released. The release profiles do not show anypronounced burst-effect, i.e. the initial release (within 4 hours) isnot more than about 10% of the incorporated dose, and more preferablynot more than about 7% of the incorporated dose.

Using the block copolymers as described above, it is possible to makemicroparticles incorporating therapeutically useful amounts ofinterferons. For example, it has been found that the polymers selectedaccording to the preferred embodiments can incorporate alfa-interferonat a content of about 0.1 to about 20 wt. % relative to the total weightof the microparticles. More preferably, the interferon content of themicroparticles is from about 0.2 to about 10 wt. %, or from 0.5 to about5 wt. %, respectively. Within these ranges, the interferon is compatiblewith the polymer matrix, with little or no tendency to aggregate. At thesame time, the concentration of the active substance is high enough toallow for a convenient administration of a relatively small volume ofmicroparticle suspension which is to be injected.

Typically, the dose of alfa-interferon per injection will be in therange from about 3 to 2,400 million international units (MIU), dependingon factors such as the state of the patient, the type and severity ofthe disease, and in particular the duration of release from themicroparticles. If the microparticles are designed to release theinterferon within about 2 or 4 weeks, respectively, the dose willnormally be in the range from about 10 to about 150 MIU. In fact, in oneof the preferred embodiments, the composition of the invention comprisesinterferon-alfa-2a, interferon-alfa-2b, or a fragment thereof, at astrength from about 10 to about 150 MIU per volume to be injected.According to a further preference, the composition has a strength in therange from about 20 to about 100 MIU per injection.

For the sake of patient comfort, the injection volume should not be veryhigh, such as no more than about 3 mL in view of the preferred route ofadministration, which is intramuscular or subcutaneous injection. In thecase of subcutaneous administration, it is more preferred that theinjection volume is not more than about 2 mL. On the other hand, highlyconcentrated injections in very small volumes are difficult to doseprecisely, for which reason it is preferred that the volume perinjection is at least about 0.1 mL, and more preferably at least about0.3 mL. The presently most preferred range is from about 0.5 mL to about2 mL.

Even though intramuscular or subcutaneous injection of the microparticlecompositions are the preferred routes of administration, it may ofcourse be possible and useful in the case of certain patients ordiseases to administer the compositions through other routes. Theseroutes are more typically parenteral routes, but may also be thepulmonary, nasal, oromucosal—such as sublingual or buccal—or otherroutes. Among the useful parenteral routes besides intramuscular andsubcutaneous injection are in particular intratumoral, intralesional,locoregional, arterial, interstitial, and intraperitoneal injections.

The microparticles and their suspension for injection are adapted forparenteral administration, which means that they are formulated andprocessed to meet the requirements of parenteral dosage forms. Suchrequirements are, for example, outlined in the major pharmacopoeias. Inone aspect, the composition, or its premixes or the kits from which thecomposition is made prior to administration, must be sterile. In anotheraspect, the excipients must be selected to be safe and tolerable forparenteral administration. In a further aspect, the compositions areformulated to be relatively isotonic (or isoosmotic), such as in theregion of about 150 to 500 mOsmol/kg, and preferably in the region ofabout 250 to 400 mOsmol/kg. Furthermore, the pH should be approximatelyin the physiological range in order to avoid pain and local intoleranceupon injection. Preferably, the pH of the composition is in the regionof about 4 to 8.5, and more preferably in the region of about 5.0 to7.5.

The microparticles are usually rendered injectable by suspending them inan appropriate, physiologically acceptable liquid carrier which ispreferably based on water, even though other biocompatible solvents suchas ethanol, glycerol, propylene glycol, polyethylene glycol, or otherorganic solvents may be present. In a more preferred embodiment, theliquid constituent of the liquid carrier is aqueous and substantiallyfree of organic solvents. On the other hand, the incorporation of otherpharmaceutical excipients may be useful or needed to optimize theproperties of the formulation, such as the tolerability, the performancein terms of drug release, and the stability. This may be true for boththe microparticles themselves and the liquid carrier. Either phase maycontain one or more additives which are physiologically tolerable.

Typically, the microparticles are resuspended in the liquid carrier toform a suspension with a solid particle content of approx. 1 to 20wt.-%, and more preferably from about 3 to 10 wt. %. The particle sizeand the viscosity of the liquid vehicle are preferably selected to allowthe injection with a relatively fine needle, such as with a 20 to 22 Gneedle. In another preferred embodiment, the particle size and theviscosity of the liquid vehicle are adapted to enable subcutaneous orintramuscular injection using a 23 to 25 G needle.

Optionally, the microparticles are designed for reconstitution usingsterile isotonic sodium chloride solution for injection.

It may be useful to stabilize the interferon with a stabilizingexcipient or combination of excipients, such as one or more salts,sugars, sugar alcohols, amino acids, peptides, proteins, polymers,surfactants, cryoprotectants, osmotic agents, buffer salts, acids, orbases. Some of these excipients may also be useful for otherpharmaceutical reasons, such as to improve the tolerability of themicroparticles or the suspension thereof. To modulate the properties ofthe polymeric carrier or improve its stability, it may be useful tofurther incorporate one or more plasticisers, pore forming agents,release-modifying agents, or antioxidants.

To avoid the agglomeration of the microparticles when suspending them inan aqueous carrier, the aqueous carrier may also contain one or morephysiologically acceptable surfactants. In fact, depending on the actualpresentation of the dosage form, a needed excipient such as a surfactantmay be incorporated either into the aqueous carrier or into a drycomposition comprising the microparticles. Selecting an appropriatesurfactant may also help to ensure that the microparticles are quicklyand easily reconstituted, such as in no more than about 3 minutes, orpreferably within about 60 seconds, and more preferably in no more thanabout 30 seconds. Examples of potentially useful surfactants includepoloxamers, polysorbates, phospholipids, and vitamin E-TPGS.

In a further embodiment, the invention provides a pharmaceutical kitcomprising the microparticles described above. In this context, apharmaceutical kit may be defined as a set of at least two compositionswhich are to be combined and used for a specific therapeutic,preventive, or diagnostic purpose. In the present case, the kitcomprises a first and a second sealed compartment which may be membersof the same or of two different primary packages. The first compartmentcomprises the composition of claim 1 in substantially dry form, whereasthe second compartment comprises an aqueous liquid carrier forreconstituting this dry composition into an injectable microparticlesuspension. Optionally, the kit contains two or more sets of each of thefirst and the second compartment.

Typically, the substantially dry composition comprised in the firstcompartment resembles one single dose to be injected, and usually alsothe second compartment will hold the volume of liquid carrier needed toreconstitute the content of the first compartment. Presently lesspreferred are compartments containing more than one dose to be injectedat one time. It is thus preferred that the content of interferon in thefirst compartment is from about 10 to about 150 MIU, and that the volumeof aqueous liquid carrier in the second compartment which can be removedwith a needle is from about 0.3 mL to about 3 mL, in particular fromabout 0.5 mL to about 2 mL.

The kit further provides a secondary package which is suitable foraccommodating the set or sets of first and second compartments.

The first and the second compartments may represent different chambersof a single device or a single primary package. For example, they may bethe two chambers of a dual chamber syringe. The advantage of prefilleddual chamber syringes is that the preparation and administration is safeand convenient as it does not require the handling of several containersunder aseptic conditions. One of the drawbacks of such syringes is thatthey are costly to provide, and may not always enable complete andreliable reconstitution.

Alternatively, the two compartments of a set may be members of twodifferent primary containers or packages. For example, the firstcompartment comprising the substantially dry microparticle compositionmay be provided in the form of a sealed bottle or vial from suitableglass or plastic, and the aqueous liquid carrier may be provided in abottle, vial, or ampoule. In a further embodiment, the first compartmentis the chamber of a syringe and the second compartment is provided as abottle, vial, or ampoule.

Optionally, one of the containers is designed as a cartridge for anauto-injecting device. Upon combining the dry composition and theaqueous liquid carrier, the ready-to-use liquid suspension is kept inthe cartridge and can be loaded into the auto-injector.

Again, it should be emphasized that either the substantially drycomposition in the first compartment or the aqueous liquid carrier, orboth, may comprise one or more further excipients, such as fillers,bulking agents, surfactants, preservatives, acids, bases, salts, sugars,sugar alcohols, amino acids, stabilizers, antioxidants, polymers,buffers, polyols, proteins such as human serum albumin, andplasticisers.

The dry composition comprising the microparticles and the aqueous liquidcarrier are adapted to yield a reconstituted suspension which issuitable for injection, i.e. which is sterile, relatively isotonic andisoosmotic, and substantially free of ingredients which are toxic whenadministered parenterally. The viscosity should be low enough to allowinjection with a needle of 17 gauge or higher, and more preferably witha needle of 20 gauge or higher, or even with a needle of 22 gauge. Asused herein, the capability of being administered refers to rheologicalproperties which allow the injection with the specified needle typewithout requiring an injection force of more than about 25 N. Morepreferably, the rheological properties are adapted, and a needle sizeselected, to enable injection with a force of no more than about 20 N,and even more preferably with an injection force of no more than about15 N, to allow the administration also to be performed by physicians,nurses, or patients who are not particularly sinewy. Of course, anotherprerequisite for such needle sizes is that the diameter of themicroparticles is sufficiently small, and the microparticles do notaggregate after reconstitution. As mentioned above, the weight averagediameter of the majority of microparticles should not be higher thanabout 200 μm, and more preferably be in the range of about 30 to about175 μm.

The microparticles of the invention may be prepared by any method knownto produce microparticles from amphiphilic polymers, such as by spraydrying, coacervation, acoustic droplet formation followed byde-solvatation, spray freeze drying etc. More preferably, however, themicroparticles are produced by an emulsion-based method which includesthe steps of (a) preparing an emulsion comprising an aqueous inner phasecomprising the active ingredient, and an organic outer phase comprisingthe biodegradable polymer and at least one organic solvent; (b)solidifying the biodegradable polymer into microparticles by removing atleast a fraction of the organic solvent from the emulsion prepared instep (a), and (c) collecting and drying the microparticles formed instep (b). The basic process design is described e.g. by J M. Bezemer etal. in J. Control Release 2000, 67 (2-3), 233-248 and 249-260, and J.Control Release 2000, 66 (2-3), 307-320, the disclosure of which isincorporated herein by reference.

Generally speaking, the microparticles are formed from an organicpolymer solution which is dispersed as droplets in an aqueous orhydrophilic phase. In order to solidify into particles, the organicsolvent must be at least partially removed from the dispersed phase.This can be accomplished by a step of solvent extraction or solventevaporation, or a combination of both. Solvent extraction means that thecontinuous aqueous phase is modified to such extent that it is capableof dissolving or extracting a substantial part of the organic solvent ofthe dispersed phase. For example, if the organic solvent has somemoderate water miscibility, a dilution or volume increase of the aqueousphase may already effect some substantial extraction of the organicphase. Alternatively, the composition of the outer phase may be modifiedby adding one or more organic solvents which are miscible with water,but which can act as cosolvents to dissolve and extract the organicsolvent of the dispersed phase. For example, ethanol, methanol, acetone,isopropyl alcohol may be used as such cosolvents.

Solvent evaporation, on the other hand, does not require the addition ofany components to directly influence the composition and properties ofthe organic phase, but makes use of the typically much higher vaporpressure of the organic solvent of the dispersed phase in comparison tothat of the aqueous phase: by applying a vacuum and or heat, the organicsolvent may be evaporated. Upon reaching a certain polymer concentrationin the organic phase, the polymer solidifies and microparticles areformed. It is important to note that any solvent evaporation of thedispersed phase will usually include the presence of the mechanism ofsolvent extraction as well.

In order to incorporate hydrophilic active compounds into themicroparticles, it may not be advisable to charge the organic phase withthe active ingredient directly. Firstly, this may lead to poorincorporation efficiency as hydrophilic compounds will partition intothe aqueous phase when the emulsion is formed. Secondly, many compoundsof interest, especially peptides and proteins such as the interferonswhich are to be incorporated according to the present invention, arerather sensitive to organic solvents and may become inactivated. It istherefore preferred that the interferon is incorporated in form of anaqueous solution which is emulsified into an organic solution of theblock copolymer to form a “water-in-oil” emulsion, which is subsequentlyemulsified into another aqueous phase to form a “water-in-oil-in-water”(w/o/w) double emulsion. When carrying out the solvent extraction orsolvent evaporation step as described above, the inner aqueous phasecomprising the interferon becomes encapsulated into the polymericmicroparticles.

One of the presently preferred organic solvents to dissolve the blockcopolymer and to provide the organic phase of the o/w-emulsion orw/o/w-double emulsion is dichloromethane. The polymer content of theorganic phase may vary according to the specific polymer composition andthe organic solvent(s) which are actually used, and may range from about1 to about 300 mg/mL. More preferably, the polymer content should be inthe range from about 50 to about 250 mg/mL, or even from about 100 toabout 150 mg/mL in the case that dichloromethane is used as solvent.

The active ingredient, i.e. the interferon, is preferably incorporatedin the form of an aqueous solution which is emulsified into the organicpolymer solution. The aqueous interferon solution may be stabilized byexcipients, such as by acids, bases, or buffer salts to achieve andmaintain a certain pH value, or by osmotic agents such as one or moresalts, sugars, sugar alcohols, amino acids etc. Some of these excipientsmay also be valuable for stabilizing effects other than related toosmolality. However, it has been found that interferon, and inparticular the alfa-interferons, can be readily incorporated using thew/o/w-double emulsion technique using a simple aqueous interferonsolution as innermost emulsion phase which contains no furtherexcipients.

The interferon content of the inner aqueous phase will obviouslyinfluence the interferon content of the microparticles, and maytherefore be selected according to the desired microparticle properties.In the case of the alfa-interferons, for example, the content may rangefrom about 1 to about 100 mg/mL, and more preferably from about 10 toabout 50 mg/mL.

The ratio of the volume of the inner aqueous phase to that of theorganic phase will also have an impact on the content of activeingredient of the microparticles. Furthermore, it may influence otherimportant properties of the particles such as their porosity and releaseprofile. Therefore, the ratio should be carefully adjusted to thedesired product characteristics in each individual case. If the featuresof the inner aqueous and the organic phases are selected according tothe preferences discussed above, a volume ratio of about 1:3 to about1:15 (inner aqueous phase:organic phase) has been found useful.According to one of the preferred embodiments, the volume ratio isselected from about 1:5 to about 1:10.

To stabilize the w/o/w-double emulsion, it may be useful to incorporateone or more stabilizers having surfactant properties into the outeraqueous phase. Useful stabilizers may be small amphiphilic moleculessuch as ionic or nonionic surfactants or detergents, or surface-activepolymers. For example, it has been found that polyvinyl alcohol is auseful additive capable of stabilizing the emulsion without having anysubstantial detrimental effects on the preparation method or on thefinal product. Useful polyvinyl alcohols may have an average molecularweight ranging from about 10,000 to about 1 million, and have a degreeof hydrolysis from about 80 to about 99%, and more preferably from about85 to about 90%. Alternatively, polyvinyl pyrrolidone or surface-activepolysaccharides may be used. The content of the stabilizer in the outerphase depends on its chemical nature, as well as on the nature andrelative volume of the dispersed organic phase. In the case of polyvinylalcohols, for example, it may range from about 0.1 to about 10 wt.-%,and more preferably from about 0.5 to about 5 wt.-%. In the case ofpolyvinyl pyrrolidone, the useful ranges are from about 1 to about 30wt.-%, and more preferably from about 5 to about 25 wt.-%.

The outer aqueous phase may also contain further excipients, such asbuffering agents, osmotic agents, or cosolvents. Cosolvents such asethanol or methanol may be used to modulate the hydrophilicity of theaqueous phase and improve any solvent extraction step of the preparationprocess. Osmotic agents may, for example, be selected from the group ofsalts, sugars, sugar alcohols, oligosaccharides, glycols, otheralcohols, and amino acids. In one of the preferred embodiments, sodiumchloride is used as an osmotic agent. It should be noted that also anybuffer system present in the outer phase will induce some osmoticpressure.

It may be useful to adjust the osmolality of the outer phase to a valuewhich is equal to, or higher than, that of the innermost aqueous phaseof the double emulsion. In this way, the osmotically driven diffusion ofwater from the outer aqueous phase to the inner aqueous phase may belargely avoided. It has been found that such diffusion process mayincrease the porosity of the microparticle formed by solvent extractionand/or solvent evaporation in a subsequent step. More preferably, theosmolality of the outer aqueous phase is adjusted to substantiallyexceed that of the innermost aqueous phase, such as by incorporatingsodium chloride at a level of about 3 to about 6 wt.-%

The relative volume of the outer phase must be selected above theminimal volume needed for the incorporation of the two other phases, andthus depends also on the nature and composition of all phases, inparticular of the organic phase and the outer aqueous phase. Above theminimal volume, the actual volume of the aqueous outer phase isimportant primarily in view of the subsequent solvent extraction and/orsolvent evaporation process. Usually, the volume of the outer aqueousphase is larger than that of the w/o-emulsion to be incorporatedtherein. For example, it may be at least twice as large as the volume ofthe w/o-emulsion. More preferably, it is about 5 to about 40 or 50 timesas large.

The preparation of the inner w/o-emulsion may be carried out usingconventional high-shear equipment, such as high-speed rotor-statordevices, e.g. of the Ultra-Turrax type, if the active ingredient isrelatively stable to shear force. To emulsify such emulsion in anaqueous phase comprising a surface-active compound, it may not be neededto apply high shear or agitation: conventional stirring equipment may besufficient. The preparation of the w/o- and w/o/w emulsions ispreferably conducted at room temperature, or at temperatures below roomtemperature, such as between about 0° C. and about 25° C., and at normalpressure. Obviously, the emulsification method used with influence theresulting average diameter and distribution of the dispersed phase, andthe size and size distribution of the microparticles. Other factors toinfluence these parameters are the compositions of the respectivephases, an in particular the nature of the organic solvent and the typeand content of the surface-active stabilizer in the outer phase.

The solidification of the polymer dissolved in the organic phase to formmicroparticles may be induced by solvent evaporation as primarymechanism. This can be accomplished by increasing the temperature of thew/o/w-double emulsion under stirring, and/or by applying a vacuum.

More preferably, however, the microparticle formation is induced by astep which includes solvent extraction. To do this, the outer phase ofthe w/o/w-double emulsion is diluted with additional aqueous solutionwhich may, optionally, be similar or even identical in its compositionto that of the outer aqueous phase. If the stabilizer content of theouter aqueous emulsion phase is sufficiently high, the aqueous solutionwhich is added for inducing the solvent extraction process may not needto contain any further stabilizer. On the other hand, it is recommendedthat the aqueous solution which is added contains an osmotically activeingredient, such as one or more salts, sugars, sugar alcohols,oligosaccharides, glycols, other alcohols, and amino acids, in order tomaintain any osmotic gradient between the inner and outer aqueous phasesof the double emulsion, and to avoid water diffusion into the innerphase. Optionally, the aqueous solution to be added may also contain acosolvent such as methanol or ethanol, or a buffering agent.

The volume of the aqueous solution which is added to the double emulsionis typically at least as large as that of the emulsion before conductingthe solvent extraction step. More preferably, the volume is from about 1to 5 times that of the double emulsion. It may be advisable to add thesolution slowly under constant stirring to avoid local inhomogeneitywithin the vessel. Optionally, the temperature may be elevated and/orsome vacuum applied to remove some of the extracted organic solvent.After the addition of the aqueous solution, stirring may be continuedfor some time to allow for a more extensive solvent extraction from theorganic phase, and perhaps also to enable the diffusion of water fromthe inner aqueous phase of the emulsion to the outer phase.

After the microparticles are solidified, they may be collected, such asby centrifugation, filtration, or sieving. Repeated centrifugation,filtration, or sieving after resuspending the microparticles in somefresh aqueous solution such as buffer should be conducted to removesubstantially all remaining organic solvents and all soluble compoundswhose presence in the microparticles is not desired. Optionally, themicroparticles may be screened to separate a desired particle sizefraction.

After washing, the microparticles may be dried for storage. A preferreddrying method is freeze drying. For example, the microparticles may befrozen in liquid nitrogen and subsequently dried under vacuum to sublimethe residual water. Usually, the drying process comprises a first dryingphase which is conducted under temperatures below 0° C. followed by asecondary drying phase at ambient or even higher temperatures.

The dried microparticles may be mixed with further optional excipientsas described above to arrive at the composition of the invention. Forexample, a powder mixture comprising the microparticles and one or moresolid-state excipients selected from the group of surfactants,resuspending agents, osmotic agents, and buffering agents may representthe composition according to claim 1. Preferably, the microparticles andthe excipients are provided in sterile form, and the mixing is conductedaseptically. Such powder mixture may be aseptically filled into bottlesor vials. As mentioned above, the bottles or vials may be combined withan aqueous liquid carrier for reconstituting the powder withpharmaceutical kits.

In a further embodiment, the composition of the invention is provided inthe form of an injectable liquid formulation. In this embodiment, theinterferon and the block copolymer are dissolved or dispersed in aliquid carrier which should be physiologically acceptable. Uponparenteral administration, the polymer solution or dispersion will forma depot in the muscle or subcutaneous tissue from which the interferonis slowly released. This embodiment is based on the discovery that theblock copolymers of the composition of the invention are indeed capableof forming macroscopic gels in a physiological environment.

Preferably, the liquid formulation is composed and adapted to be capableof forming a gel after injection. A gel may be defined by virtue of itsrheological properties. As used herein, a gel is a semisolid materialwhich behaves like a solid upon the exertion of low shear force, andlike a viscous fluid when the shear force exceeds a threshold which isdefined as the yield point. In other words, a gel is a system with afinite, usually rather small, yield stress.

Injectable gels and in-situ-forming gels as controlled release dosageforms have been described by A. Hatefi et al., J. Control. Rel. 80(2002), 9-28, which document is incorporated herein by reference. Thereare several general approaches to the formulation of an injectable gel,most of which are based on the use of gel-forming polymeric carriers.For example, certain polymers may form gels which are responsive tocertain conditions of the environment, such as pH or temperature. Forexample, sol-gel systems have been described which are present as sols(viscous, colloidal liquid solutions) at a relatively low pH or at roomtemperature. When injected, the pH is slowly buffered by physiologicalfluids to a more neutral value, resulting in solidification and gelformation. In a temperature-responsive system, the temperature risesafter injection to a physiological level, leading to the gelling of thesystem.

More preferably, however, the injectable solution comprises anon-aqueous, biocompatible organic solvent, or cosolvent, which in vitroprovides a liquid solution or suspension, but which, after injection,slowly diffuses away from the block copolymer, which is insoluble butcapable of gel formation in an aqueous environment.

The organic solvent or cosolvent may be selected from those organicsolvents which are capable of dissolving the block copolymer(s) andwhich may be considered biocompatible in view of the intendedadministration volume and frequency. Examples of such solvents includebenzyl alcohol, benzyl benzoate, diacetin, tributyrin, triethyl citrate,tributyl citrate, acetyl triethyl citrate, acetyl tributyl citrate,triethylglycerides, triethyl phosphate, diethyl phthalate, diethyltartrate, polybutene, glycerine, ethylene glycol, polyethylene glycol,octanol, ethyl lactate, propylene glycol, propylene carbonate, ethylenecarbonate, butyrolactone, ethylene oxide, propylene oxide,N-methyl-2-pyrrolidone, 2-pyrrolidone, glycerol formal, methyl acetate,ethyl acetate, methyl ethyl ketone, dimethylformamide, dimethylsulfoxide, tetrahydrofuran, caprolactam, decylmethylsulfoxide, oleicacid, 1-dodecylazacycloheptan-2-one, and mixtures thereof.

In one of the preferred embodiments, the non-aqueous solvent is one ormore members of the group consisting of DMSO, NMP, benzyl alcohol,tetrahydrofuran, ethyl acetate, and benzyl benzoate.

The block copolymer content of the liquid injectable composition istypically from about 5 wt.-% to about 60 wt.-%, primarily depending onthe precise polymer or polymers which are actually used. Morepreferably, the polymer content is from about 15 to about 45 wt.-%.

Particularly suitable block copolymers for carrying out this aspect ofthe invention comprise a relatively high average PEGT content, such asfrom about 70 to about 98 wt.-%, and more preferably from about 75 toabout 95 wt.-%. Presently most preferred are block copolymers having anaverage PEGT content from about 80 to about 90 wt.-%.

The average molecular weight of the PEG segments of the PEGT blocks istypically from about 300 to about 6,000, and more preferably from about600 to about 2,000.

Optionally, the composition may comprise more two or more one blockcopolymers which differ in their PEGT content, in the molecular weightof the PEG segments, or in both of these parameters. In a preferredembodiment, the composition comprises one or two block copolymers.

Again, the composition may comprise one or more further excipients, suchas one or more cosolvents, surfactants, preservatives, acids, bases,salts, sugars, sugar alcohols, amino acids, stabilizers, antioxidants,osmotic agents, and polymers. The rationale for incorporating any ofthese excipients may be the same as discussed further above in thecontext of microparticle-based compositions. Alternatively, an excipientmay serve any function typically associated with liquid injectableformulations.

Typically, the volume of the liquid formulation is from about 0.3 toabout 3 mL per dose to be injected, and more preferably from about 0.5to about 2 mL.

The injectable formulation of the invention is typically designed forintramuscular or subcutaneous injection. These routes of administrationnecessitate certain quality-related properties which are generallyrequired for parenteral products, such as sterility. Thus, it ispreferred that the injectable liquid formulation is sterile and meetsall requirements of parenteral dosage forms as specified in the majorpharmacopoeias, such as the current United States Pharmacopoeia (USP).

The injectable liquid formulation can be prepared by dissolving theblock copolymer(s) the in the non-aqueous biocompatible solvent,optionally at an elevated temperature. The active compound, i.e. theinterferon, may be added to this polymer solution as a dry powder, suchas a lyophilized powder, under stirring. Preferably, the interferon isnot incorporated in form of an aqueous solution in order to avoid thepresence of water in the formulation.

As a further embodiment, the invention provides the composition of claim1 in form of a macroscopic solid implant. An implant may be defined as asolid, substantially dry dosage form which is different frommicroparticles in that an implant typically contains a single dose ofactive ingredient within a single dosage unit, or within only a fewunits. Usually, the largest dimension of an implant is in the range ofseveral millimeters or more, whereas microparticles are administered asmultiple units and have dimension below the millimeter scale.

In one of the preferred embodiments, the implant is shaped as a rod.This is particularly advantageous in terms of a “less invasive”administration which largely avoids tissue injury. Furthermore,polymeric rod-shaped articles may be prepared efficiently by meltextrusion followed by cutting the extrudate into rods. To carry out suchextrusion, the block copolymer(s), the interferon, and the furtherexcipients should be provided in dry powder or granule form and mixedhomogeneously. Subsequently, the mixture is fed into an extruder, suchas a single or twin screw extruder, and extruded into a coherent solidstrand which is then cut into individual rods.

The composition of the block copolymer may be selected as discussed inthe context of the microparticles further above. A type of excipientwhich may be particularly useful in implants is a plasticizer, which maydecrease the melting range or glass transition point of the polymer(s)to temperature which does not have a negative impact on the stability ofthe incorporated interferon. Potentially useful plasticizers includeglycerol, propylene glycol, and polyethylene glycol.

Irrespective of whether the composition is provided in the form of amicroparticle-based formulation, an injectable liquid or gel, or a solidimplant, the pharmaceutical use is the preparation of a drug product forthe management of diseases and conditions which may be treated, or whoseprogression may be prevented or decelerated, by the administration of aninterferon, and most preferably by the administration of analfa-interferon. Examples of these diseases and conditions include acuteand chronic hepatitis B, acute and chronic hepatitis C, hairy cellleukaemia, acute and chronic myelogenous leukaemia, multiple myeloma,follicular lymphoma, carcinoid tumor, malignant melanoma, condylomaacuminata, SARS, and Kaposi's sarcoma, such as AIDS-related Kaposi'ssarcoma.

The composition of the invention provides the advantage overconventional interferon formulations for injection that the frequency ofinjection may be greatly reduced by virtue of its controlled releasecharacteristics, such as to one injection every 2 or 4 weeks instead ofseveral injections per week. As a consequence, patient comfort andcompliance are increased, and the costs associated with frequentinjections potentially reduced. With regard to other polymericcontrolled release systems for injection or implantation, the presentinvention provides excellent compatibility with interferons, improvedrelease control without burst effect, dose dumping, or autocatalyticpolymer degradation and erosion. Furthermore, the delivery systems ofthe invention are physiologically well-tolerated, without producing anysignificant carrier-related side effects.

Without wishing to be bound by a particular theory, the good releasebehavior of the delivery systems of the invention seems to be related tothe fact that the active compound is released primarily throughdiffusion, and not through erosion as in the case of many of thepresently known poly(lactide)- and/or -(glycolide)-based deliverysystems. Using amphiphilic block copolymers, there is no autocatalyticpolymer degradation involved in the release process. In contrast to theknown delivery systems, the block copolymers do not produce an acidicmicroenvironment which is hostile to sensitive biological compounds. Onthe other hand, the hydrophilic blocks of the block copolymers probablyprovide a hydrophilic microenvironment which enhances the in-situstability of such sensitive biological compounds. In particular, itseems that interferons—especially interferons of the alpha family—arestabilized in a non-aggregated state in the microenvironment provided bythe amphiphilic block copolymers in the carrier systems of theinvention.

Especially in the case of microparticles, it is also believed that therelatively low porosity of the particles formed from the blockcopolymers is one of the causes of the low burst effect observed in thecompositions of the invention.

Further embodiments, applications and advantages of the invention willbecome obvious from the following non-limiting examples, or may beeasily derived by persons skilled in the field of drug delivery on thebasis of this description.

Example 1 Preparation of a W/O/W Double Emulsion ContainingInterferon-Alfa-2b

Non-glycosylated, recombinant interferon-alfa-2b (IFN-α-2b), a proteincomposed of 165 amino acids, having a molecular weight of approx. 19,000Da and an isoelectric point of about 6.0, was obtained in form of anaqueous solution with a protein concentration of approx. 10 mg/mL. Blockcopolymer of 80 wt.-% PEGT and 20 wt.-% PBT with PEG segments having anaverage molecular weight of 1,500 was obtained from IsoTis, Bilthoven,The Netherlands. A solution of 1 g polymer in 7 mL of dichloromethanewas prepared. To prepare a w/o-emulsion, 1 mL of the IFN-alfa-2bsolution was added to the polymer solution under stirring, followed byultra turrax homogenization at 19,000 rpm for approx. 30 seconds.

Two different w/o/w/-double emulsions were prepared by pouring twow/o-emulsions—prepared as described above—separately into 50 mL of (a)an aqueous PBS buffer containing 4% PVA (w/v) (MW approx. 130,000,degree of hydrolysis approx. 87%), or (b) an aqueous sodium chloridesolution (5% w/v) also containing 4% PVA (w/v) under stirring at 700rpm.

Example 2 Preparation of Microparticles by Solvent Extraction andEvaporation

The double emulsions prepared according to Example 1 were furtherprocessed to prepare microparticles. To each of the two doubleemulsions, 100 mL of aqueous PBS buffer was slowly added undercontinuous stirring at 700 rpm. The added PBS solution led to anexpansion of the outer aqueous phase of the double emulsions.Subsequently, stirring was continued for approx. 1 hour to extract themajor part of the dichloromethane into the outer aqueous phase, and tothe solidification of the polymer in the organic phase. Subsequently,the solidified microparticles were centrifuged at 2,500 rpm and at roomtemperature. The supernatant was discarded, and the pellet wasresuspended in fresh PBS buffer to be centrifuged again. The procedurewas repeated three times. Finally, the microparticles were frozen inliquid nitrogen and freeze dried over approx. 12-24 hours. Theencapsulation efficiency was determined to be about 85% for themicroparticles from the w/o/w double emulsion whose outer phasecontained 5% sodium chloride, and approx. 25% for the other batch. Themicroparticles were examined by electron microscopy (SEM) and found tobe roughly spherical and predominantly in the size range of about 50 toabout 120 μm.

Example 3 Interferon-Alfa-2b Release from Microparticles In Vitro

For testing their release behavior, approx. 15 mg of each batch of themicroparticles prepared according to Example 2 were weighed in 1.5 mLflasks in triplicate. To each flask, 1 mL of PBS buffer was added. Theflasks were kept in a water bath at 37° C. At sampling times, themicroparticles were centrifuged at 1,000 rpm for 2 minutes at roomtemperature. Samples of 700 μl were withdrawn and replaced by fresh PBSbuffer. The amount of IFN-alfa-2b in each sample was determined with aMicro bichinchonic acid total protein assay.

It was found that both batches clearly demonstrated sustained releasecharacteristics. The batch obtained from the w/o/w double emulsion whoseouter phase contained 5% sodium chloride showed an initial burst effectof less than about 10%, while the other batch had a burst effect ofabout 20%. Both batches released 50% of their interferon content withinabout 3-4 days, and 75% within about 7-8 days. After 14 days, approx.85-90% of the incorporated dose was released.

Example 4 Preparation of a Composition Comprising MicroparticlesIncorporating Truncated IFN-Alpha-2b

A microparticle-comprising composition was prepared as follows, usingaseptic conditions. A quantity of 6 g of a sterile block copolymer of 77wt.-% PEGT and 23 wt.-% PBT with PEG segments having an averagemolecular weight of 1,500 was weighed and dissolved in 54 g steriledichloromethane. The organic polymer solution was combined with 5.5 mLof a sterile aqueous solution comprising a mixture of N-terminallytruncated INF-alpha-2b molecules having, in average, a length of about158 amino acid residues, a specific activity of about 0.25 to 0.35 MIUper μg, and an interferon concentration of about 10 mg/mL. Anultraturrax device was used to obtain a homogenous water-in-oilemulsion.

Subsequently, the emulsion was combined under stirring with 445 g of asterile aqueous solution of polyvinyl alcohol (4% w/v) which alsocontained sodium chloride (5% w/v). Thereby, a w/o/w double emulsion wasobtained in which the polyvinyl alcohol solution formed the outeraqueous phase.

In the next step, microparticles were formed and hardened by the removalof solvent from the organic phase, which was accomplished by acombination of solvent extraction and solvent evaporation. Some solventextraction was conducted by the addition of sterile PBS buffer to thecontinuous phase of the double emulsion, and another portion ofdichloromethane was evaporated by blowing sterile nitrogen at a flowrate of about 5-10 L/min over the surface of the double emulsion forabout 24 hours.

The microparticles were collected and washed with sterile mannitolsolution (26.7 g/L) and resuspended in an appropriate volume of mannitolsolution to adjust the osmolality to a physiologically tolerable valueand to enable adequate cake formation upon lyophilization. Aliquots ofthe suspension were filled into sterile glass vials and freeze dried,resulting in white lyophilisates. The vials were closed with plasticstoppers and aluminum caps.

Analytical testing showed that the number average diameter of themicroparticles was about 83 μm, and from the interferon content it wasconcluded that the encapsulation efficiency was higher than about 90%.The residual dichloromethane was substantially below 600 ppm. Electronmicrographs of the microparticles revealed little porosity; inparticular, most of the particles had no pores having a diameter of morethan about 2-5 μm.

Example 5 In Vivo Testing of Microparticles Comprising a TruncatedIFN-Alpha-2b

Microparticle-comprising compositions prepared in analogy to example 4were tested for their in vivo performance in hamsters and monkeys. Thesolid lyophilized compositions were suspended in a sterile aqueoussolution of sodium carboxymethyl cellulose (0.1% w/v), optionallyfurther containing mannitol to adjust the osmolality of the liquidphase. The amounts of aqueous solution were calculated, based on thecontent of active ingredient and the dose to be administered, to yieldinjection volumes of 0.5 to 1.0 mL per single administration. Each often hamsters received a dose of 0.99 mg/kg of the active compoundadministered by s.c. injection every 7 days, and another group of tenhamsters received 3.46 mg/kg every 7 days. Serum samples were obtainedfrom the animals at selected intervals, which samples were stored infrozen form and later analyzed for their interferon content. All animalsappeared to tolerated the treatment well.

Based on the serum profiles, the in vivo release profiles of the activecompound were calculated. In a separate experiment, the in vitro releaseprofiles were determined as described in Example 3. A comparison of thein vivo and in vitro release profiles showed that there was a goodcorrelation between the respective profiles, both in shape and induration of release, and that the in vitro release behavior appears tobe an excellent predictor of the in vivo performance of thecompositions. There was no significant burst effect in vivo or in vitro.

FIG. 1 shows the calculated average in vivo release profiles of the lowdose hamster group, the high dose group, and the respective in vitrorelease profile, normalized to 100% total release.

In another series of experiments, samples of the same composition wereadministered subcutaneously to male and female monkeys. The dose ofactive ingredient was 180 μg per animal, and the same reconstitutionliquid was used to disperse the composition to a volume of 0.5 to 1.0 mlper injection. Starting from the time of injection, serum samples wereobtained at selected time intervals over a period of 14 days. Again, theserum concentrations were used to calculate the in vivo releaseprofiles, which were then compared to the in vitro release profilesdetermined from other samples of the same batch of the compositionaccording to the method described in Example 3.

In result, the correlation between the respective release profiles wasremarkable. Both in vitro and in vivo the composition appeared torelease its interferon content steadily over a period of 14 days,without any substantial burst release.

FIG. 2 shows the calculated average in vivo release profile and therespective in vitro release profile, normalized to 100% total release.

Example 6 Purity of Released Interferon

Samples of the released interferon obtained from in vitro releasetesting as described in Examples 4 and 5 were analyzed byhigh-performance size-exclusion chromatography to determine the fractionof interferon which was release in monomeric form. Remarkably, it wasfound that less than 1% of the release active compound was in the formof dimers or larger aggregates, even though alpha-interferons are knownto agglomerate easily. Thus, the microparticle composition hasapparently contributed to substantial stabilization of the interferon.

Example 7 Preparation of Film Composition Incorporating Block Copolymerand Interferon-Beta

A quantity of 0.5 g of a block copolymer of 80 wt.-% PEGT and 20 wt.-%PBT with PEG segments having an average molecular weight of 2,000 wasweighed and dissolved in 3.5 mL dichloromethane. 1.94 mg of lyophilizedinterferon-beta were dispersed in the solution using an ultraturrax. Thedispersion was cast onto glass plates, using an adjustable filmapplicator. After evaporation of the dichloromethane, films wereobtained and stripped from the glass plate. The films were dried furtherin the fume hood for some hours.

Samples of about 1.77 cm² were cut out from the films and incubated inan aqueous acetate buffer solution pH 3.5 (1 ml) at 37° C. in a shakingwater bath. After every 24 hours of incubation, the whole volume ofrelease medium was refreshed and the samples were further incubated.Aliquots of the withdrawn buffer were used for HP-SEC analysis, whichrevealed that about 83% of the incorporated beta-interferon was releasedin monomeric, non-aggregated form. The duration of release depended onthe thickness of the films.

Example 8 Preparation and Release Properties of Self-Gelling BlockCopolymer Solution Containing IFN-Alfa-2a

Interferon-alfa-2a and a PEGT/PBT block copolymer containing 85 wt.-%PEGT with PEG segments having an average molecular mass of about 1,000were obtained in dry form. The polymer was dissolved in a mixture ofbenzyl benzoate and benzyl alcohol (98:2) at a concentration of 20wt.-%. The interferon was added in powder form at a concentration of 4wt.-% and thoroughly mixed with the polymer solution. The resultingmixture was filled into a syringe with a needle and injected into PBSbuffer solution at 37° C. Upon injection, an irregular gel slowlyprecipitated. The gel was kept at 37° C. under continuous agitation.Samples were withdrawn at appropriate time intervals and replaced byfresh PBS buffer solution. The samples were analyzed for their contentof IFN-alfa-2b and confirmed a release time of more than about 14 days(90% release).

1. A method for the preparation of a pharmaceutical compositioncomprising microparticles including one or more active compoundsselected from the group of interferons and one or more biodegradableblock copolymers constructed from poly(ethylene glycol) terephthalate(PEGT) and poly(butylene terephthalate) (PBT), wherein the averagemolecular weight of the polyethylene glycol (PEG) is from about 1,000 toabout 2,000 daltons, wherein the block copolymer comprises from about 50to about 95 wt.% PEGT, and wherein the composition comprises a dose ofat least 20 MIU of interferon that is released over a period of about 10days to about 1 month, comprising the steps of: (a) preparing anemulsion comprising (aa) an aqueous inner phase comprising the one ormore active compounds, and (ab) an organic outer phase comprising theone or more biodegradable block copolymer and at least one organicsolvent; (b) solidifying the one or more biodegradable block copolymerinto microparticles by removing at least a fraction of the organicsolvent from the emulsion prepared in step (a); (c) collecting anddrying the microparticles formed in step (b) wherein step (b) is carriedout by emulsifying the emulsion prepared in step (a) in a coherentaqueous phase to obtain a w/o/w-double emulsion, wherein the coherentaqueous phase comprises an osmotic agent, wherein the osmolality of thecoherent aqueous phase is higher than that of the aqueous inner phase.2. The method of claim 1, wherein the osmotic agent is a salt.
 3. Themethod of claim 2, wherein the salt is sodium chloride.
 4. The method ofclaim 3, wherein the sodium chloride is present at a level of about 3 toabout 6 wt. %.
 5. The method of claim 1, wherein the interferon isselected from the group of alpha-interferons.
 6. The method of claim 2,wherein the interferon is selected from the group of alpha-interferons.7. The method of claim 3, wherein the interferon is selected from thegroup of alpha-interferons.
 8. The method of claim 4, wherein theinterferon is selected from the group of alpha-interferons.